Modified transketolase and use thereof

ABSTRACT

The present invention relates to a improved process for the biotechnological production of compounds for which ribose-5-phosphate, ribulose-5-phosphate or xylulose-5-phosphate is biosynthetic precursor like riboflavin (vitamin B 2 ), FAD, FMN, pyridoxal phosphate (vitamin B 6 ), guanosine, GMP, adenosine, AMP. The invention further pertains to the generation of the organism producing those compounds. It furthermore relates to the generation of mutated transketolases that allow normal growth on glucose but reduced growth on gluconate when introduced into the production strains and to polynucleotides encoding them.

This application is the U.S. national phase under 35 USC 371 of Int'lApplication No. PCT/EP2006/010270, filed 25 Oct. 2006, which designatedthe U.S. and claims priority to European Patent Application No.05023813.8, filed 2 Nov. 2005; the entire contents of each of which arehereby incorporated by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 2, 2011, isnamed 4662-788.txt and is 67.628 bytes in size.

The present invention provides modified transketolase enzymes.Microorganisms synthesizing one of the modified transketolases insteadof the wild type transketolase are prototroph for aromatic amino acidsand impaired in using carbon sources that are assimilated via thepentose phosphate pathway. The modified enzymes and polynucleotidesencoding the same can be used in the fermentation process for substancesthat use ribose-5-phosphate, ribulose-5-phosphate, orxylulose-5-phosphate as substrate for the biosynthesis such as e.g.riboflavin, riboflavin precursors, flavin mononucleotide (FMN), flavinadenine dinucleotide (FAD), and derivatives thereof. They also can beused for the production of pyridoxal phosphate (vitamin B₆), guanosineand adenosine and derivatives of these nucleotides.

BACKGROUND OF THE INVENTION

Riboflavin (vitamin B₂) is synthesized by all plants and manymicroorganisms but is not produced by higher animals. Because it is aprecursor to coenzymes such as flavin adenine dinucleotide and flavinmononucleotide that are required in the enzymatic oxidation ofcarbohydrates, riboflavin is essential to basic metabolism. In higheranimals, insufficient riboflavin can cause loss of hair, inflammation ofthe skin, vision deterioration, and growth failure.

The enzymes required catalyzing the biosynthesis of riboflavin fromguanosine triphosphate (GTP) and ribulose-5-phosphate are encoded byfour genes (ribG, ribB, ribA, and ribH) in B. subtilis. These genes arelocated in an operon, the gene order of which differs from the order ofthe enzymatic reactions catalyzed by the enzymes. For example, GTPcyclohydrolase II, which catalyzes the first step in riboflavinbiosynthesis, is encoded by the third gene in the operon, ribA. The ribAgene also encodes a second enzymatic activity, i.e.,3,4-dihydroxy-2-butanone 4-phosphate synthase (DHBPS), which catalyzesthe conversion of ribulose-5-phosphate to the four-carbon unit3,4-dihydroxy-2-butanone 4-phosphate (DHBP). Deaminase and reductase areencoded by the first gene of the operon, ribG. The penultimate step inriboflavin biosynthesis is catalyzed by lumazine synthase, the productof the last rib gene, ribH. Riboflavin synthase, which controls the laststep of the pathway, is encoded by the second gene of the operon, ribB.The function of the gene located at the 3′ end of the rib operon is, atpresent, unclear; however, its gene product is not required forriboflavin synthesis.

Transcription of the riboflavin operon from the ribP1 promoter iscontrolled by an attenuation mechanism involving a regulatory leaderregion located between ribP1 and ribG. ribO mutations within this leaderregion result in deregulated expression of the riboflavin operon.Deregulated expression is also observed in strains containing missensemutations in the ribC gene. The ribC gene has been shown to encode theflavin kinase/FAD synthase of B. subtilis (Mack, M., et al., J.Bacteriol., 180:950-955, 1998). Deregulating mutations reduce theflavokinase activity of the ribC gene product resulting in reducedintracellular concentrations of flavin mononucleotide (FMN), theeffector molecule of the riboflavin regulatory system.

Engineering of riboflavin production strains with increased productionrates and yields of riboflavin has been achieved in the past in a numberof different ways. For instance, (1) classical mutagenesis was used togenerate variants with random mutations in the genome of the organism ofchoice, followed by selection for higher resistance to purine analogsand/or by screening for increased production of riboflavin. (2)Alternatively, the terminal enzymes of riboflavin biosynthesis, i.e.,the enzymes catalyzing the conversion of guanosine triphosphate (GTP)and ribulose-5-phosphate to riboflavin, were over-expressed, resultingalso in a higher flux towards the target product. The metabolic fluxinto and through a biosynthetic pathway, e.g. the riboflavinbiosynthetic pathway, is determined by the specific activities of therate-limiting enzymes of this particular pathway and by theintracellular concentrations of the substrates for these enzymes. Onlyat or above saturating substrate concentrations an enzyme can operate atits maximal activity. The saturating substrate concentration is acharacteristic feature for each enzyme. For example, the metabolic fluxinto the riboflavin pathway may be increased or kept at a high level bykeeping the intracellular concentrations of ribulose-5-phosphate aboveor as close as possible to the saturating substrate concentration of the3,4-dihydroxy-2-butanone 4-phosphate synthase, a presumed rate limitingenzyme for the riboflavin biosynthetic pathway. High intracellularconcentrations of ribulose-5-phosphate may, for example, be reached bypreventing or interfering with drainage of ribulose-5-phosphate into thecentral metabolism via the non-oxidative part of the pentose phosphatepathway.

A key enzyme in the non-oxidative part of the pentose phosphate pathwayis the transketolase enzyme, which catalyzes the reversible conversionof ribose-5-phosphate and xylulose-5-phosphate toseduheptulose-7-phosphate and glyceraldehyde-3-phosphate. In addition,transketolase catalyzes also the conversion of fructose-6-phosphate andglyceraldehyde-3-phosphate to xylulose-5-phosphate anderythrose-4-phosphate (Kochetov, G. A. 1982, Transketolase from yeast,rat liver, and pig liver, Methods Enzymol., 90:209-23).

It has previously been reported that transketolase deficient Bacillussubtilis strains carrying knock-out mutations in the transketolaseencoding gene produces ribose, which accumulates in the fermentationbroth (De Wulf, P., and E. J. Vandamme. 1997. Production of D-ribose byfermentation, Appl. Microbiol. Biotechnol. 48:141-148; Sasajima, K., andYoneda, M. 1984, Production of pentoses by microorganisms. Biotechnol.and Genet. Eng. Rev. 2: 175-213). Obviously, increased intracellular C5carbon sugar pools can be reached in transketolase knock-out mutants upto a level that exceeds the physiological requirements of the bacteriaand leads to secretion of excess ribose.

As mentioned above, transketolase catalysed reactions are also requiredto produce erythrose-4-phosphate, from which the three proteinogenicaromatic amino acids are derived. Therefore, transketolase deficientmicroorganisms are auxotroph for these amino acids. They can only growif these amino acids or their biosynthetic precursors, for instanceshikimic acid, can be supplied via the cultivation medium.

In addition to the unfavorable auxotrophy for aromatic amino acids orshikimic acid, transketolase-deficient B. subtilis mutants show a numberof severe pleiotropic effects like very slow growth on glucose, adefective phosphoenolpyruvate-dependent phosphotransferase system,deregulated carbon catabolite repression, and altered cell membrane andcell wall composition (De Wulf, P., and E. J. Vandamme. 1997).

An other transketolase-deficient riboflavin secreting B. subtilis strainwas described by Gershanovich et al. (Gershanovich V N, Kukanova A I a,Galushkina Z M, Stepanov A I (2000) Mol. Gen. Mikrobiol. Virusol.3:3-7).

Furthermore, U.S. Pat. No. 6,258,554 B1 discloses a riboflavinoverproducing Corynebacterium glutamicum strain in which transketolaseactivity is deficient. It can be noted form the disclosure of the U.S.Pat. No. 6,258,554 B1 that the deficiency in transketolase activity andthe resulting amino acid auxotrophy was essential for the improvedriboflavin productivity, since a prototrophic revertant producedriboflavin in amounts similar to a C. glutamicum strain with a wild-typetransketolase background.

These disadvantages, i.e. auxotrophy for aromatic amino acids andfurther pleiotropic effects discussed above, make a transketolasedeficient mutant a less preferable production strain for stableindustrial processes, such as, e.g. the industrial production ofriboflavin within such strain.

SUMMARY OF THE INVENTION

It is in general an object of the present invention to provide atransketolase mutant strain which is modified in such a way that thecatalytic properties of the modified transketolase allowing higherintracellular ribulose-5-phosphate and ribose-5-phosphate concentrationsthan those of the non-modified transketolase, but which does not havethe disadvantages of the transketolase-deficient strains mentionedabove.

Surprisingly, it has now been found that by genetically altering amicroorganism such as for instance B. subtilis, by replacing thewild-type gene by a mutated gene encoding a modified transketolase thatallows some residual flux through the pentose phosphate pathway byhaving modulated specific activities, the production of a fermentationproduct such as e.g. riboflavin can be significantly improved withoutloosing the prototrophic properties.

The present invention relates to modified transketolases, polynucleotidesequences comprising a gene that encodes a modified transketolase withproperties described above, a host cell which has been transformed bysuch a polynucleotide sequence, and a process for the biotechnologicalproduction of a fermentation product such as for instance riboflavin, ariboflavin precursor, FMN, FAD, pyridoxal phosphate or one or morederivatives thereof based on a host cell in which the wild-typetransketolase gene has been stably replaced by a polynucleotide codingfor the mutated transketolase.

As a first step to isolate mutants, in which the wild-type transketolaseis replaced by one of such modified transketolases, a deletion mutantmay be generated that is auxotroph for the proteinogenic aromatic aminoacids and cannot grow with carbon sources assimilated via the pentosephosphate pathway, e.g. gluconate. The transketolase deletion mutant maythen be transformed with a mixture of DNA fragments encoding varioustransketolase mutants. Prototrophic transformants may be isolated, fromwhich those are selected which show a reduced growth rate on gluconate.Mutants isolated according to this method may synthesize modifiedtransketolase enzymes that allow sufficient erythrose-4-phosphatebiosynthesis to prevent auxotrophic growth, but act as a bottle neck forassimilation of gluconate. In addition, the undesired pleiotropiceffects typically observed with B. subtilis transketolase deletionmutants may be prevented. U.S. Pat. No. 6,258,554 B1 indicates thattogether with the reversion of the auxotrophic to the prototrophicgrowth riboflavin secreting C. glutamicum transketolase mutants losttheir ability to produce more riboflavin than a similar straincontaining a wild-type transketolase gene. As shown in the examples ofthe present invention, prototrophic B. subtilis transketolase mutantsisolated as outlined above unexpectedly produced more riboflavin thanthe transketolase wild-type parent strain, whereas a transketolasedeletion mutant had partly lost their riboflavin productioncapabilities.

Methods for the introduction of mutations into DNA fragments are wellknown in the art. Transketolase mutants can be generated for instance byprotein engineering using one of the available 3D structures of e.g. theyeast transketolase (Lindqvist, Y., G. Schneider, U. Ermler, and M.Sundstrom. 1992. Three-dimensional structure of transketolase, athiamine diphosphate dependent enzyme, at 2.5 A resolution. Embo. J.11:2373-9) for selecting suitable positions of the amino acid sequenceor by random mutagenesis. The selection process in both cases may bedone as described above. The modified transketolase—when it is used assubstitution for the wild-type transketolase—exhibits catalyticproperties, i.e. modulated specific activities, which allow the growthof a host cell on a carbon source that is metabolized exclusively by thepentose phosphate pathway (for example gluconate) with a reduced growthrate in comparison to a host cell containing the wild-typetransketolase. These properties result in higher intracellularribulose-5-phosphate and ribose-5-phosphate concentrations and aresidual flux through the pentose phosphate pathway, so that sufficienterythrose-4-phosphate can be produced to prevent auxotrophic growth.

“Wild-type enzyme” or “wild-type transketolase” which can be used forthe present invention may include any transketolase as defined abovethat is used as starting point for designing mutants according to thepresent invention. The wild-type transketolase may be of eukaryotic orprokaryotic, preferably fungal or bacterial origin, in particularselected from Escherichia, Bacillus, Corynebacterium, Saccharomyces,Eremothecium, Candida or Ashbya, preferably from E. coli, B. subtilis,B. licheniformis, B. halodurans, S. cerevisiae, E. gossypii, C. flarerior A. gossypii or any transketolase having an amino acid sequence whichis homologous to an amino acid sequence as shown in FIG. 1. Mostpreferably the transketolase is from B. subtilis. “Homologous” refers toa transketolase that is at least about 50% identical, preferably atleast about 60% identical, more preferably at least about 70%, 80%, 85%,90%, 95% identical, and most preferably at least about 98% identical toone or more of the amino acid sequences as shown in FIG. 1. “Wild-type”in the context of the present invention may include both transketolasesequences derivable from nature as well as variants of synthetictransketolase enzymes (as long as they are homologous to any one of thesequences shown in FIG. 1), The terms “wild-type transketolase” and“non-modified transketolase” are used interchangeably herein.

The term “% identity”, as known in the art, means the degree ofrelatedness between polypeptide or polynucleotide sequences, as the casemay be, as determined by the match between strings of such sequences.“Identity” can be readily determined by known methods, e.g., with theprogram BESTFIT (GCG Wisconsin Package, version 10.2, Accelrys Inc.,9685 Scranton Road, San Diego, Calif. 92121-3752, USA) using thefollowing parameters: gap creation penalty 8, gap extension penalty 2(default parameters).

A “mutant”, “mutant enzyme”, “mutated enzyme” or “mutant transketolase”or a “modified transketolase” as used herein means any variant derivablefrom a given wild-type enzyme/transketolase (according to the abovedefinition) according to the teachings of the present invention and,when used for replacing the wild-type gene of a host organism/cellshould have an effect on the growth on e.g. gluconate and/or ribose. Forthe scope of the present invention, it is not relevant how the mutant(s)are obtained; such mutants may be obtained, e.g., by site-directedmutagenesis, saturation mutagenesis, random mutagenesis/directedevolution, chemical or UV mutagenesis of entire cells/organisms, etc.These mutants may also be generated, e.g., by designing synthetic genes,and/or produced by in vitro (cell-free) translation. For testing ofspecific activity, mutants can may be (over-) expressed by methods knownto those skilled in the art. The terms “mutant transketolase”, “modifiedtransketolase” or “mutated transketolase” are used interchangeablyherein.

“Host cell” is a cell capable of producing a given fermentation productand containing the wild-type transketolase, or a nucleic acid encodingthe modified transketolase according to the invention. Suitable hostcells include cells of microorganisms.

As used herein, the term “specific activity” denotes the reaction rateof the wild-type and mutant transketolase enzymes under properly definedreaction conditions as described in Kochetov (Kochetov, G. A. 1982.Transketolase from yeast, rat liver, and pig liver. Methods Enzymol90:209-23). The “specific activity” defines the amount of substrateconsumed and/or product produced in a given time period and per definedamount of protein at a defined temperature. Typically, “specificactivity” is expressed in μmol substrate consumed or product formed permin per mg of protein. Typically, μmol/min is abbreviated by U (=unit).Therefore, the unit definitions for specific activity of μmol/min/(mg ofprotein) or U/(mg of protein) are used interchangeably throughout thisdocument. It is understood that in the context of the present invention,specific activity must be compared on the basis of a similar, orpreferably identical, length of the polypeptide chain.

Many mutations may change a wild-type transketolase in such a way thatgrowth on gluconate is affected as described above.

It is an object of the present invention to provide a modifiedtransketolase having the properties defined above, wherein the aminoacid sequence of the modified transketolase contains at least onemutation when compared with the amino acid sequence of the correspondingnon-modified transketolase.

The at least one mutation may be an addition, deletion and/orsubstitution.

Preferably, the at least one mutation is at least one amino acidsubstitution wherein a given amino acid present in the amino acidsequence of the non-modified transketolase is replaced with a differentamino acid in the amino acid sequence of the modified transketolase ofthe invention. The amino acid sequence of the modified transketolase maycontain at least one amino acid substitution when compared with theamino acid sequence of the corresponding non-modified transketolase.Particularly, a modified transketolase as of the present inventioncontains at least one mutation on an amino acid position whichcorresponds to amino acid position 357 of the B. subtilis transketolaseamino acid sequence as depicted in SEQ ID NO:2.

In further embodiments, the modified transketolase contains at leasttwo, at least three, at least four or at least five substitutions whencompared with the amino acid sequence of the correspondingtransketolase. For example, the modified transketolase contains one toten, one to seven, one to five, one to four, two to ten, two to seven,two to five, two to four, three to ten, three to seven, three to five orthree to four amino acid substitutions when compared with the amino acidsequence of the corresponding non-modified transketolase.

In a preferred embodiment of the invention the non-modifiedtransketolase is obtainable from Bacillus, preferably B. subtilis, asdepicted in SEQ ID NO:2. The corresponding DNA sequence is shown in SEQID NO:1. The modified transketolase contains at least one mutation onposition 357 of SEQ ID NO:2, leading to a modified transketolase havingthe above described properties.

The at least one amino acid substitution in the non-modifiedtransketolase located on a position corresponding to amino acid 357 asshown in SEQ ID NO:2 may be selected from substitution R357H, R357A,R357S, R357N, R357T, R357K, R3571, R357V, R357G, and R357L.

In a particularly preferred embodiment, the mutated transketolaseconsists of one substitution which affects the amino acid positioncorresponding to amino acid position 357 of the amino acid sequence asshown in SEQ ID NO:2 and which may be selected from substitution R357H,R357A, R357S, R357N, R357T, R357K, R3571, R357V, R357G, and R357L.

In an other preferred embodiment, the modified transketolase contains atleast two amino acid substitutions when compared with the amino acidsequence of the corresponding non-modified transketolase, wherein atleast one mutation corresponding to amino acid position 357 of the aminoacid sequence as shown in SEQ ID NO:2 and which may be selected fromsubstitution R357H, R357A, R357S, R357N, R357T, R357K, R3571, R357V,R357G, and R357L.

The amino acid present in the non-modified transketolase is preferablyarginine at position 357. The amino acid in the sequence of thenon-modified transketolase may be changed to histidine, alanine, serine,asparagine, lysine, threonine, leucine, glycine, isoleucine or valine atposition 357. Preferably, the substitution at the amino acid positioncorresponding to position 357 of the sequence as shown in SEQ ID NO: 2consists of the replacement of arginine with histidine, arginine withalanine, arginine with serine, arginine with leucine, arginine withlysine, arginine with asparagine, arginine with threonine, arginine withglycine, arginine with isoleucine, arginine with valine.

The modified transketolase of the invention may comprise foreign aminoacids, preferably at its N- or C-terminus. “Foreign amino acids” meanamino acids which are not present in a native (occurring in nature)transketolase, preferably a stretch of at least about 3, at least about5 or at least about 7 contiguous amino acids which are not present in anative transketolase. Preferred stretches of foreign amino acids includebut are not limited to “tags” that facilitate purification of therecombinantly produced modified transketolase. Examples of such tagsinclude but are not limited to a “His₆” tag (SEQ ID NO: 31), a FLAG tag,a myc tag, and the like. For calculation of specific activity, thevalues need to be corrected for these additional amino acids (see alsoabove).

In another embodiment the modified transketolase may contain one ormore, e.g. two, deletions when compared with the amino acid sequence ofthe corresponding non-modified transketolase. Preferably, the deletionsaffect N- or C-terminal amino acids of the corresponding non-modifiedtransketolase and do not significantly reduce the functional properties,e.g., the specific activity, of the enzyme.

The invention further relates to a polynucleotide comprising anucleotide sequence which codes for a modified transketolase accordingto the invention. “Polynucleotide” as used herein refers to apolyribonucleotide or polydeoxyribonucleotide that may be unmodified RNAor DNA or modified RNA or DNA. Polynucleotides include but are notlimited to single- and double-stranded DNA, DNA that is a mixture ofsingle- and double-stranded regions, single- and double-stranded RNA,and RNA that is a mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that may be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions. The term “polynucleotide” includes DNA or RNA that comprisesone or more unusual bases, e.g., inosine, or one or more modified bases,e.g., tritylated bases.

The polynucleotide of the invention can easily be obtained by modifyinga polynucleotide sequence which codes for a non-modified transketolase.Examples of such polynucleotide sequences encoding non-modifiedtransketolase enzymes include but are not limited to the amino acidsequences of FIG. 1. Preferably, the non-modified transketolase isoriginated from Bacillus, in particular B. subtilis, more preferred is apolynucleotide encoding a non-modified transketolase as depicted in SEQID NO:2.

Methods for introducing mutations, e.g., additions, deletions and/orsubstitutions into the nucleotide sequence coding for the non-modifiedtransketolase include but are not limited to site-directed mutagenesisand PCR-based methods.

DNA sequences of the present invention may be constructed starting fromgenomic or cDNA sequences coding for transketolase enzymes known in thestate of the art, as are available from, e.g., Genbank (Intelligenetics,California, USA), European Bioinformatics Institute (Hinston Hall,Cambridge, GB), NBRF (Georgetown University, Medical Centre, WashingtonD.C., USA) and Vecbase (University of Wisconsin, Biotechnology Centre,Madison, Wis., USA) or from the sequence information disclosed in FIG. 1by methods of in vitro mutagenesis [see e.g. Sambrook et al., MolecularCloning, Cold Spring Harbor Laboratory Press, New York]. Anotherpossibility of mutating a given DNA sequence which may also be suitablefor the practice of the present invention is mutagenesis by using thepolymerase chain reaction (PCR). DNA as starting material may beisolated by methods known in the art and described, e.g., in Sambrook etal. (Molecular Cloning) from the respective strains/organisms. It is,however, understood that DNA encoding a transketolase to beconstructed/mutated in accordance with the present invention can also beprepared on the basis of a known DNA sequence, e.g. by construction of asynthetic gene by methods known in the art (as described, e.g., in EP747483).

Once complete DNA sequences of the present invention have been obtained,they can be integrated into vectors or directly introduced into thegenome of a host organism by methods known in the art and described in,e.g., Sambrook et al. (s.a.) to (over-) express the encoded polypeptidein appropriate host systems. However, a man skilled in the art knowsthat also the DNA sequences themselves can be used to transform thesuitable host systems of the invention to get (over-) expression of theencoded polypeptide.

In a preferred embodiment the present invention provides

-   (i) a DNA sequence which codes for a modified transketolase carrying    at least one mutation as defined above and which hybridizes under    standard conditions with any of the DNA sequences of the specific    modified transketolase enzymes, for example which hybridizes with    the DNA sequences according to SEQ ID NO:1, or-   (ii) a DNA sequence which codes for a modified transketolase    carrying at least one mutation as defined above but, because of the    degeneracy of the genetic code, does not hybridize but which codes    for a polypeptide with exactly the same amino acid sequence as a DNA    sequence which hybridizes under standard conditions with any of the    DNA sequences of the specific modified transketolase enzymes of the    present invention, or-   (iii) a DNA sequence which is a fragment of such modified DNA    sequences which maintains the activity properties of the polypeptide    of which it is a fragment.

“Standard conditions” for hybridization mean in the context of thepresent invention the conditions which are generally used by a manskilled in the art to detect specific hybridization signals and whichare described, e.g. by Sambrook et al., “Molecular Cloning”, secondedition, Cold Spring Harbor Laboratory Press 1989, New York, orpreferably so-called stringent hybridization and non-stringent washingconditions or more preferably so-called stringent hybridization andstringent washing conditions a man skilled in the art is familiar withand which are described, e.g., in Sambrook et al. (s.a.). A specificexample of stringent hybridization conditions is overnight incubation(e.g., 15 hours) at 42° C. in a solution comprising: 50% formamide,5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml ofdenatured, sheared salmon sperm DNA, followed by washing thehybridization support in 0.1×SSC at about 65° C.

In another preferred embodiment the invention further provides a DNAsequence which can be obtained by the so-called polymerase chainreaction method (“PCR”) by PCR primers as shown in FIG. 2, designed onthe basis of the specifically described DNA sequences.

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably purified tohomogeneity.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living microorganism is not isolated, but thesame polynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition and still be isolated inthat such vector or composition is not part of its natural environment.

An isolated polynucleotide or nucleic acid as used herein may be a DNAor RNA that is not immediately contiguous with both of the codingsequences with which it is immediately contiguous (one on the 5′-end andone on the 3′-end) in the naturally occurring genome of the organismfrom which it is derived. Thus, in one embodiment, a nucleic acidincludes some or all of the 5′-non-coding (e.g., promoter) sequencesthat are immediately contiguous to the coding sequence. The term“isolated polynucleotide” therefore includes, for example, a recombinantDNA that is incorporated into a vector, into an autonomously replicatingplasmid or virus, or into the genomic DNA of a prokaryote or eukaryote,or which exists as a separate molecule (e.g., a cDNA or a genomic DNAfragment produced by PCR or restriction endonuclease treatment)independent of other sequences. It also includes a recombinant DNA thatis part of a hybrid gene encoding an additional polypeptide that issubstantially free of cellular material, viral material, or culturemedium (when produced by recombinant DNA techniques), or chemicalprecursors or other chemicals (when chemically synthesized). Moreover,an “isolated nucleic acid fragment” is a nucleic acid fragment that isnot naturally occurring as a fragment and would not be found in thenatural state.

As used herein, the term isolated polypeptide refers to a polypeptidethat is substantially free of other polypeptides. An isolatedpolypeptide is preferably greater than 80% pure, more preferably greaterthan 90% pure, even more preferably greater than 95% pure, mostpreferably greater than 99% pure. Purity may be determined according tomethods known in the art, e.g., by SDS-PAGE and subsequent proteinstaining. Protein bands can then be quantified by densitometry. Furthermethods for determining the purity are within the level of ordinaryskill.

As mentioned above, the modified transketolases and the correspondingpolynucleotides of the invention may be utilized in the geneticengineering of a suitable host cell to make it better and more efficientin the fermentation process for substances that use ribose-5-phosphate,ribulose-5-phosphate, or xylulose-5-phosphate as substrate for thebiosynthesis. The presence of said modified transketolase within asuitable host cell may result in higher intracellularribulose-5-phosphate and ribose-5-phosphate concentrations and aresidual flux through the pentose phosphate pathway within saidrecombinant host, so that sufficient erythrose-4-phosphate can beproduced to prevent auxotrophic growth.

Appropriate host cells are for example fungi, like Aspergilli, e.g.Aspergillus niger or Aspergillus oryzae, or like Trichoderma, e.g.Trichoderma reesei, or Ashbya, e.g. Ashbya gossypii, or Eremothecium,e.g. Eremothecium ashbyii, or yeasts like Saccharomyces, e.g.Saccharomyces cerevisiae, or Candida, like Candida flareri, or Pichia,like Pichia pastoris, or Hansenula polymorpha, e.g. H. polymorpha (DSM5215). Bacteria which can be used are, e.g., Bacilli as, e.g., Bacillussubtilis or Streptomyces, e.g. Streptomyces lividans. E. coli whichcould be used are, e.g., E. coli K12 strains, e.g. M15 or HB 101.

Thus, the present invention relates to a microorganism wherein theactivity of a transketolase is modified in such a way that themicroorganism is capable of growing on a carbon source that ismetabolized exclusively by the pentose phosphate pathway (for examplegluconate) with a reduced growth rate in comparison to a host cellcontaining the wild-type transketolase. It is in general possible tointroduce an obtained transketolase mutant originating from a certainorganism e.g. B. subtilis in the same organism again and now used as ahost cell or to introduce any obtained mutant into any other relevanthost cells.

As used herein, the term “growth rate” denotes to the following:Bacterial cells reproduce by dividing in two. If growth is not limited,doubling continues at a constant rate so both the number of cells andthe rate of population increase doubles with each consecutive timeperiod. For this type of exponential growth, plotting the naturallogarithm of cell number against time (preferably in hours) produces astraight line. The slope of this line is the specific growth rate of theorganism, which is a measure of the number of divisions per cell perunit time. In food, bacteria cannot grow continuously as the amount ofnutrient available will be finite and waste products will accumulate. Inthese conditions growth curves tend to be sigmoid.

It is an object of the present invention to provide a recombinant hostcell wherein the growth rate of said recombinant host cell (e.g.microorganism) according to the present invention carrying a modifiedtransketolase on a carbon source that is metabolized exclusively by thepentose phosphate pathway, in particular gluconate, is less than 100%when compared to the wild-type organism. In particular, the growth ratemay be reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% oreven 90% and more compared to the growth rate of a wild-type organism.Preferably, the growth rate reduction is between 10% and 90%, morepreferably between 20% and 80%, still more preferably between 25% and75% compared to a cell containing a wild-type transketolase gene.

Especially, the invention relates to a geneticallyengineered/recombinantly produced host cell (also referred to asrecombinant cell or transformed cell) in which the wild-typetransketolase gene has been replaced by a modified transketolase geneencoding an enzyme that allows a slightly or non-reduced growth on acarbon source that is not exclusively metabolized by the pentosephosphate pathway, but shows a clearly reduced growth rate when theorganism grows on a carbon source that is metabolized exclusively by thepentose phosphate pathway. Such genetically engineered host cells showan improvement of the yield of the fermentation product and of theefficiency of the production process with the advantages that undesiredauxotrophic growth and pleiotropic effects can be prevented.

The invention further relates to a process for producing a host cellscapable of expressing a transketolase according to the invention,comprising the steps of

-   (i) generating mutated transketolases displaying modulated    activities in comparison to the respective wild-type enzyme, i.e.-   a) providing a polynucleotide encoding a first or non-modified    transketolase with catalytic properties that should be adapted;-   b) introducing one or more mutations into the polynucleotide    sequence such that the mutated polynucleotide sequence encodes a new    or modified transketolase which contains at least one amino acid    mutation when compared to the first transketolase wherein the at    least one amino acid mutation may be at amino acid corresponding to    position 357 of the amino acid sequence as shown in SEQ ID NO: 2;-   c) optionally inserting the mutated polynucleotide in a vector or    plasmid;-   (ii) replacing the wild-type transketolase(s) of the host cell by a    transketolase variant from the same organisms or another organism    that allows normal or slightly reduced growth on a carbon source    that is not exclusively metabolized by the pentose phosphate    pathway, but shows an effect on the growth rate when the organism    grows on a carbon source that is metabolized exclusively by the    pentose phosphate pathway, i.e.-   a) replacing the wild-type transketolase of a suitable wild-type    host cell without altering the regulatory sequences of the gene;-   b) determining the growth rate on gluconate in minimal medium and    comparing it to the wild-type host strain; and-   c) selecting transketolase mutants that allow a growth rate on    gluconate which is less than 100% of the wild-type strain.

The invention further relates to a method for the production ofsubstances that are secondary products of ribose-5-phosphate,ribulose-5-phosphate, or xylulose-5-phosphate comprising:

-   a) culturing a genetically engineered/recombinantly produced host    cell in which the wild-type transketolase gene has been replaced by    a modified transketolase gene encoding an enzyme that allows a    slightly or non-reduced growth on a carbon source that is not    exclusively metabolized by the pentose phosphate pathway, but which    shows a reduced growth rate when the organism grows on a carbon    source that is metabolized exclusively by the pentose phosphate    pathway, in a suitable medium under conditions that allow expression    of the modified transketolase; and-   b) separating the fermentation product from the medium.

The “fermentation product” as used herein may be any product produced bya suitable host cell as defined above the biosynthesis of which usesribose-5-phosphate, ribulose-5-phosphate, or xylulose-5-phosphate assubstrate. Examples of such fermentation products include but are notlimited to riboflavin, riboflavin precursors, flavin mononucleotide(FMN), flavin adenine dinucleotide (FAD) and derivatives thereof,pyridoxal phosphate (vitamin B₆), guanosine, adenosine and derivativesof these nucleotides.

“Riboflavin precursor” and “derivatives of riboflavin, FMN or FAD” inthe context of this invention shall include any and all metabolite(s)requiring ribulose-5-phosphate or ribulose-5-phosphate as anintermediate or substrate in their (bio-) synthesis. In the context ofthis patent application, it is irrelevant whether such (bio-) synthesispathways are natural or non-natural (i.e., pathways not occurring innature, but engineered biotechnologically). Preferably, the synthesispathways are biochemical in nature. Riboflavin precursors andderivatives of riboflavin, FMN or FAD include but are not limited to:DRAPP; 5-amino-6-ribosylamino-2,4 (1H,3H)-pyrimidinedione-5′-phosphate;2,5-diamino-6-ribitylamino-4 (3H)-pyrimidinone-5′-phosphate;5-amino-6-ribitylamino-2,4 (1H,3H)-pyrimidinedione-5′-phosphate;5-amino-6-ribitylamino-2,4 (1H,3H)-pyrimidinedione;6,7-dimethyl-8-ribityllumazine (DMRL); and flavoproteins. The term“riboflavin” also includes derivatives thereof, such as e.g.riboflavin-5-phosphate and salts thereof, such as e.g. sodiumriboflavin-5-phospate.

The polynucleotides, polypeptides, recombinant host cells and methodsdescribed herein may be used for the biotechnological production ofeither one or more of the fermentation products as defined above.

Methods of genetic and metabolic engineering of suitable host cellsaccording to the present invention are known to the man skilled in theart. Similarly, (potentially) suitable purification methods for e.g.riboflavin, a riboflavin precursor, FMN, FAD, pyridoxal phosphate or oneor more derivatives thereof are well known in the area of fine chemicalbiosynthesis and production.

It is understood that a method for biotechnological production of afermentation product such as for instance riboflavin, a riboflavinprecursor, FMN, FAD, pyridoxal phosphate or one or more derivativesthereof according to the present invention is not limited towhole-cellular fermentation processes as described above, but may alsouse, e.g., permeabilized host cells, crude cell extracts, cell extractsclarified from cell remnants by, e.g., centrifugation or filtration, oreven reconstituted reaction pathways with isolated enzymes. Alsocombinations of such processes are in the scope of the presentinvention. In the case of cell-free biosynthesis (such as withreconstituted reaction pathways), it is irrelevant whether the isolatedenzymes have been prepared by and isolated from a host cell, by in vitrotranscription/translation, or by still other means.

Fermentation media must contain suitable carbon substrates. Suitablesubstrates may include but are not limited to monosaccharides such asglucose or fructose, oligosaccharides such as lactose or sucrose,polysaccharides such as starch or cellulose or mixtures thereof andunpurified mixtures from renewable feedstocks. It is contemplated thatthe source of carbon utilized in the present invention may encompass awide variety of carbon containing substrates and will only be limited bythe choice of organism.

The various embodiments of the invention described herein may becross-combined.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will now be illustrated in more detail by thefollowing non-limiting examples. These examples are described withreference to the Figures.

FIG. 1-1, FIGS. 1-2, and FIGS. 1-3 shows—as already mentionedabove—examples of polynucleotide sequences which code for a non-modifiedtransketolase, and

FIG. 2 shows a set of primers, in which tkt 1S is SEQ ID NO:3, tkt 1ASis SEQ ID NO:13, tkt 1ASohne is SEQ ID NO:6, tkt 2S is SEQ ID NO:4, tkt2AS is SEQ ID NO:4, tkt 3S is SEQ ID NO:9, tkt 4S is SEQ ID NO:10, tkt5S is SEQ ID NO:11, tkt 6S is SEQ ID NO:12, tkt 357AS is SEQ ID NO:14,tkt Rec 1S is SEQ ID NO:27, tkt Rec 1AS is SEQ ID NO:28, tkt Rec 2S isSEQ ID NO:29, tkt Rec 2AS is SEQ ID NO:30, tkt 357A-S is SEQ ID NO:17,tkt 357N-S is SEQ ID NO:15, tkt 357K-S is SEQ ID NO:18, tkt 357Q-S isSEQ ID NO:16, tkt 357S-S is SEQ ID NO:19, tkt 357T-S is SEQ ID NO:20,tkt 357H-S is SEQ ID NO:21, tkt 357V-S is SEQ ID NO:22, tkt 3571-S isSEQ ID NO:23, tkt 357L-S is SEQ ID NO:24, tkt 357M-S is SEQ ID NO:25,tkt 357G-S is SEQ ID NO:26, rpi MutS is SEQ ID NO:5.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In particular, FIG. 1 shows multiple sequence alignment calculated bythe program clustalW (1.83) of the transketolase amino acid sequencesfrom Escherichia coli (TKT_ECOLI), Bacillus subtilis (TKT_BACSU),Bacillus licheniformis (TKT_BACLD), Bacillus halodurans (TKT_BACHD),Corynebacterium glutamicum (TKT_CORGL), Saccharomyces cerevisiae(TKT_YEAST), and Ashbya gossypii (TKT_ASHGO). Positions that arehomologous/equivalent to the amino acid residue 357 of the B. subtilistransketolase that are discussed in one of the following examples are inbold letters. The numbering used for those positions is done accordingto the B. subtilis wild-type amino acid sequence. This type of alignmentcan be done with CLUSTAL or PILEUP using standard parameters. As shown,the amino acid sequence of transketolases is highly conserved. Inparticular, in all transketolases shown and in much more transketolasesnot shown, arginine 357 (numbering according to the B. subtilistransketolase) is conserved. Therefore, the type of experiment using theconcepts and mutations reported here can also be done with othertransketolases having an arginine at a position homologous to position357 of the amino acid sequence of B. subtilis transketolase like Ashbyagossypii for improving the production of riboflavin, riboflavinderivatives or compounds having ribose-5-phosphate,xylulose-5-phosphate, or ribulose-5-phosphate as a precursor. It is alsopossible to replace an original transketolase gene of an organism by aB. subtilis transketolase mutant gene mutated at position 357 with orwithout adaptation of the DNA sequence to the new organism. It is notessential that the transketolase mutant genes originate from an organismin which it is going to be introduced. The practical steps required foranother host organism are published and known to an expert in the fieldand outlined somewhere else.

EXAMPLE 1 Isolation of Genomic DNA from Bacillus subtilis

gDNA was prepared using the DNeasy Tissue Kit from Qiagen (QIAGEN GmbH,QIAGEN Str. 1, 40724 Hilden, Germany) according to the description ofthe supplier. 1 ml of a 3 ml overnight culture of B. subtilis in VYliquid medium (Becton Dickinson, Sparks, Md. 21152, USA) incubated at37° C. (250 rpm) was used as source for the bacteria cells. At the end,the gDNA was eluted in 200 μl of AE buffer (supplied with the Kit).

EXAMPLE 2 Amplification of the Transketolase Gene from Bacillus subtilis

gDNA from B. subtilis PY79 (P. Youngman, J. Perkins, and R. Losick(1984), Construction of a cloning site near one end of Tn917 into whichforeign DNA may be inserted without affecting transposition in Bacillussubtilis or expression on the transposon-borne erm gene. Plasmid 12:1-9;see Example 1) was used for amplification of the tkt gene. According tothe genomic DNA sequence, the tkt gene contains one Eco RI site insideof its coding sequence (SEQ ID NO: 1). Since the Eco RI restriction siteis generally used for cloning into E. coli expression vectors such aspQE80 (QIAGEN GmbH, QIAGEN Str. 1, 40724 Hilden, Germany), the site wasdeleted by replacing C315 by a T, which is a silent mutation changingthe phenylalanine codon from TTC to TTT. For this, two separate PCRs Aand B were performed. The following PCR conditions were used for PCR A:2 μM of primer tkt 1S (according to SEQ ID No: 3, see also FIG. 2) andtkt 2AS (according to SEQ ID No: 4, FIG. 2), 0.2 mM of each nucleotide(ATP, GTP, TTP, CTP), 2.5 U of a proof-reading DNA polymerase(Stratagene, Gebouw California, 1101 CB Amsterdam Zuidoost, TheNetherlands), 100 ng genomic DNA (Example 1) in the appropriate bufferas supplied together with the DNA polymerase.

Temperature regulation was as follows:

-   Step 1: 3 min at 95° C.-   Step 2: 30 sec at 95° C.-   Step 3: 30 sec at 52° C.-   Step 4: 30 sec at 72° C.-   Step 5: 5 min at 72° C.

Steps 2 to 4 were repeated 35-times.

PCR B was done under the following conditions: 2 μM of primer tkt 2S(according to SEQ ID No: 8, FIG. 2) and tkt 1AS (according to SEQ ID No:13, FIG. 2), 0.2 mM of each nucleotide (ATP, GTP, TTP, CTP), 2.5 U of aproof-reading DNA polymerase (Stratagene, Gebouw California, 1101 CBAmsterdam Zuidoost, The Netherlands), 100 ng genomic DNA (Example 1) inthe appropriate buffer as supplied together with the DNA polymerase.

Temperature regulation was as follows:

-   Step 1: 3 min at 95° C.-   Step 2: 30 sec at 95° C.-   Step 3: 30 sec at 52° C.-   Step 4: 2 min at 72° C.-   Step 5: 5 min at 72° C.

Steps 2 to 4 were repeated 35-times.

The two PCR products A and B were purified by Agarose gelelectrophoresis and a following extraction out of the gel using theMinElute Gel Extraction Kit from Qiagen (QIAGEN GmbH, QIAGEN Str. 1,40724 Hilden, Germany). Using the overlapping region of PCR products Aand B, it was possible to assemble them by a third PCR: 2 μM of primerRpi MutS (according to SEQ ID No: 5, FIG. 2) and tkt 1ASohne (accordingto SEQ ID No: 6, FIG. 2), 0.2 mM of each nucleotide (ATP, GTP, TTP,CTP), 2.5 U of a proof-reading DNA polymerase (Stratagene, GebouwCalifornia, 1101 CB Amsterdam Zuidoost, The Netherlands), 100 ng of PCRproduct A and PCR product B in the appropriate buffer as suppliedtogether with the DNA polymerase.

-   Step 1: 3 min at 95° C.-   Step 2: 30 sec at 95° C.-   Step 3: 30 sec at 53° C.-   Step 4: 2.5 min at 72° C.-   Step 5: 5 min at 72° C.

Steps 2 to 4 were repeated 35-times.

The PCR products were purified with the help of the Qiagen PCRpurification Kit (QIAGEN GmbH, QIAGEN Str. 1, 40724 Hilden, Germany) andeluted in 50 μl elution buffer. The PCR product was confirmed by an EcoRI digestion. For further confirmation, it was sequenced with theprimers tkt IS, tkt 2S, tkt 2AS, tkt 3S (according to SEQ ID No: 9, FIG.2), tkt 4S (according to SEQ ID No: 10, FIG. 2), tkt 5S (according toSEQ ID No: 11, FIG. 2), tkt 6S (according to SEQ ID No: 12, FIG. 2), tkt1AS.

EXAMPLE 3 Construction of Tkt Mutants

The 3D structure of the yeast transketolase was available together witha selection of mutations that showed influence on substrate binding ofthe yeast transketolase (Nilsson, U., L. Meshalkina, Y. Lindqvist, andG. Schneider. 1997. At position R359 (number 357 in the B. subtilistransketolase), the original arginine was replaced by nearly all otheramino acids. The construction of the mutants was basically done asdescribed in example 1. An amino acid sequence alignment comprising thetransketolases from yeast, B. subtilis and from other organisms is shownin FIG. 1.

Using the Eco RI-free tkt gene as template (Example 2), the mutationswere introduced as already described for the deletion of the Eco RIsite: The following PCR conditions were used for PCR A and B: 2 μM ofprimer Rpi MutS (A) or tkt 357nnn-S (B) and tkt 357AS (A) (according toSEQ ID No: 14, FIG. 2) or tkt 1ASohne (B), 0.2 mM of each nucleotide(ATP, GTP, TTP, CTP), 2.5 U of a proof-reading DNA polymerase(Stratagene, Gebouw California, 11011 CB Amsterdam Zuidoost, TheNetherlands), 100 ng of the Eco RI-free tkt gene (Example 2) in theappropriate buffer as supplied together with the DNA polymerase. In thecase of PCR B the sense primer was chosen according to the amino acidthat was introduced: tkt 357N-S (according to SEQ ID No: 15, FIG. 2) forasparagine, tkt 357Q-S (according to SEQ ID No: 16, FIG. 2) forglutamine, tkt 357A-S (according to SEQ ID No: 17, FIG. 2) for alanine,tkt 357K-S (according to SEQ ID No: 18, FIG. 2) for lysine, tkt 357S-S(according to SEQ ID No: 19, FIG. 2) for serine, tkt 357T-S (accordingto SEQ ID No: 20, FIG. 2) for threonine, tkt 357H-S (according to SEQ IDNo: 21, FIG. 2) for histidine, tkt 357V-S (according to SEQ ID No: 22,FIG. 2) for valine, tkt 3571-S (according to SEQ ID No: 23, FIG. 2) forIsoleucine, tkt 357L-S (according to SEQ ID No: 24, FIG. 2) for leucine,tkt 357M-S (according to SEQ ID No: 25, FIG. 2) for methionine, and tkt357G-S (according to SEQ ID No: 26, FIG. 2) for the introduction ofglycine at position 357 of the B. subtilis transketolase.

Temperature regulation was as follows:

-   Step 1: 3 min at 95° C.-   Step 2: 30 sec at 95° C.-   Step 3: 30 sec at 52° C.-   Step 4: 60 sec at 72° C.-   Step 5: 5 min at 72° C.

Steps 2 to 4 were repeated 35-times.

The two PCR products A and B were purified by Agarose gelelectrophoresis and a following extraction out of the gel using theMinElute Gel Extraction Kit from Qiagen (QIAGEN GmbH, QIAGEN Str. 1,40724 Hilden, Germany). Assembling of PCR product

A and B was done in a third PCR: 2 μM of primer Rpi MutS and tkt1ASohne, 0.2 mM of each nucleotide (ATP, GTP, TTP, CTP), 2.5 U of aproof-reading DNA polymerase (Stratagene, Gebouw California, 1101 CBAmsterdam Zuidoost, The Netherlands), 100 ng of PCR product A and PCRproduct B in the appropriate buffer as supplied together with the DNApolymerase.

-   Step 1: 3 min at 95° C.-   Step 2: 30 sec at 95° C.-   Step 3: 30 sec at 53° C.-   Step 4: 2.5 min at 72° C.-   Step 5: 5 min at 72° C.

Steps 2 to 4 were repeated 35-times.

The PCR products of the transketolase were purified with the Qiagen PCRpurification Kit (QIAGEN GmbH, QIAGEN Str. 1, 40724 Hilden, Germany) andeluted in 50 μl elution buffer. The PCR products were used for thetransformation of B. subtilis.

EXAMPLE 4 Construction of a Transketolase-Deficient B. subtilis Strain

For the marker free introduction of a mutated transketolase gene intothe original tkt locus of the B. subtilis genome, atransketolase-deficient strain was constructed. Two DNA fragmentsobtained by PCR comprising base pair 452 to 1042 and base pair 1562 to2001 of the B. subtilis transketolase gene (SEQ ID NO: 2) were combinedwith the neomycin resistance gene cassette (M. Itaya, K. Kondo, and T.Tanaka. 1989. A neomycin resistance gene cassette selectable in a singlecopy state in the Bacillus subtilis chromosome. Nucleic Acids Res17:4410). The following PCR conditions were used for PCR A: 2 μM ofprimer tkt Rec1S (according to SEQ ID No: 27, FIG. 2) and tkt Rec1AS(according to SEQ ID No: 28, FIG. 2), 0.2 mM of each nucleotide (ATP,GTP, TTP, CTP), 2.5 U of a proof-reading DNA polymerase (Stratagene,Gebouw California, 1101 CB Amsterdam Zuidoost, The Netherlands), 100 ngof the amplified tkt gene of Example 2 in the appropriate buffer assupplied together with the DNA polymerase.

Temperature regulation was as follows:

-   Step 1: 3 min at 95° C.-   Step 2: 30 sec at 95° C.-   Step 3: 30 sec at 52° C.-   Step 4: 30 sec at 72° C.-   Step 5: 5 min at 72° C.

Steps 2 to 4 were repeated 30-times.

PCR B was done under the following conditions: 2 μM of primer tkt Rec 2S(according to SEQ ID No: 29, FIG. 2) and tkt Rec 2AS (according to SEQID No: 30, FIG. 2), 0.2 mM of each nucleotide (ATP, GTP, TTP, CTP), 2.5U of a proof-reading DNA polymerase (Stratagene, Gebouw California, 1101CB Amsterdam Zuidoost, The Netherlands), 100 ng of the amplified tktgene of Example 2 in the appropriate buffer as supplied together withthe DNA polymerase.

Temperature regulation was as follows:

-   Step 1: 3 min at 95° C.-   Step 2: 30 sec at 95° C.-   Step 3: 30 sec at 52° C.-   Step 4: 2 min at 72° C.-   Step 5: 5 min at 72° C.

Steps 2 to 4 were repeated 30-times.

The two PCR products A and B were purified by Agarose gelelectrophoresis and a following extraction out of the gel using theMinElute Gel Extraction Kit from Qiagen (QIAGEN GmbH, QIAGEN Str. 1,40724 Hilden, Germany). Due to the overlapping regions of the two PCRproducts A and B with the sequence of the neomycin resistance cassette,it is possible to assemble them by a third PCR: 2 μM of primer tkt Rec1S and tkt Rec 2AS, 0.2 mM of each nucleotide (ATP, GTP, TTP, CTP), 2.5U of a proof-reading DNA polymerase (Stratagene, Gebouw California, 1101CB Amsterdam Zuidoost, The Netherlands), 100 ng of PCR product A, 100 ngof PCR product B, and 100 ng of neomycin resistance cassette in theappropriate buffer as supplied together with the DNA polymerase.

-   Step 1: 3 min at 95° C.-   Step 2: 30 sec at 95° C.-   Step 3: 30 sec at 55° C.-   Step 4: 2.5 min at 72° C.-   Step 5: 5 min at 72° C.

Steps 2 to 4 were repeated 35-times.

Five assembling PCRs were pooled and purified with the Qiagen PCRpurification Kit (QIAGEN GmbH, QIAGEN Str. 1, 40724 Hilden, Germany) andeluted in 50 μl elution buffer. The correct PCR product was confirmed byagarose gel electrophoresis and used for transformation of B. subtilisPY79. Preparation of competent B. subtilis cells was done according toKunst et al., 1988 (F. Kunst, M. Debarbouille, T. Msadek, M. Young, C.

Mauel, D. Karamata, A. Klier, G. Rapoport, and R. Dedonder. 1988.Deduced polypeptides encoded by the Bacillus subtilis sacU locus sharehomology with two-component sensor-regulator systems. J Bacteriol 170:5093-101). 2 ml MNGE+Bacto Casamino Acid (CAA) (9 ml MN-medium (13.6 g/lK₂HPO₄, 6.0 g/l KH₂PO₄, 0.88 g/l sodium citrate*2H₂O), 1 ml glucose(20%), 40 μl potassium glutamate (40%), 50 μl, ammonium iron(III)citrate (2.2 mg/l, freshly prepared), 100 μl tryptophan (8 mg/l), 30 μlMgSO₄ (1 M), +/−50 μl Bacto Casamino Acid (20%, Becton Dickinson AG,Postfach, CH-4002 Basel, Switzerland) were inoculated with a singlecolony and incubated overnight at 37° C. and 250 rpm. This culture wasused to inoculate 10 ml MNGE+CAA (start OD₅₀₀ nm of 0.1) and wasincubated at 37° C. under shaking (250 rpm) until it reached an OD₅₀₀ nmof 1.3. The culture was diluted with the same volume of MNGE w/o CAA andwas incubated for another hour. After a centrifugation step (10 min,4000 rpm, 20° C.), the supernatant was decanted into a sterile tube. Thepellet was re-suspended in ⅛ of the kept supernatant. 300 μl of cellswere diluted in 1.7 ml MN (1×), 43 μl glucose (20%) and 34 μl MgSO₄ (1M). 10 and 20 μl of the prepared PCR product was added to 400 μl of thediluted competent cells and shaked for 30 min at 37° C. 100 μlexpression mix (500 μl 5% yeast extract (Becton Dickinson AG, Postfach,CH-4002 Basel, Switzerland), 125 μl CAA (20%), 1/100 of the finalantibiotic concentration (2 μg/ml neomycin), if used for selection, and750 μl sterile bidest. water) were added and the cells were shaked for 1h at 37° C. At the end, the cells were spun down, suspended in 200 μl ofthe supernatant and plated onto TBAB plates (Becton Dickinson AG,Postfach, CH-4002 Basel, Switzerland) containing 2 μg/ml neomycin.

Two transformants were grown in VY medium (5 g/l yeast extract (BectonDickinson AG, Postfach, CH-4002 Basel, Switzerland), 25 g/l vealinfusion broth (Sigma)). From one of the transformants, designatedBS3402, the genomic DNA was isolated as described in Example 1 and thecorrect replacement of the transketolase DNA fragment from base pair1043 to 1561 by the neomycin gene cassette was confirmed by a standardPCR using tkt Rec 1 S and tkt Rec 2AS as primers. As expected for atransketolase deletion mutant, the strain could not grow on ribose orgluconate as sole carbon source and required all three aromatic aminoacids or shikimic acid for growth.

EXAMPLE 5 Transformation of the Transketolase-Deficient B. subtilisStrain BS3402 with the Genes of the Transketolase Variants

0.5 and 1 μg DNA of the amplified transketolase gene and its variants(Example 2 and 3) were used to transform BS3402 as described in Example4. Positive colonies were identified by growth on minimal medium (2 g/lglucose and sorbitol in SMS-medium (2 g/l (NH₄)₂SO₄, 14 g/l K₂HPO₄, 6g/l KH₂PO₄, 1 g/l tri-sodium citrate, 0.2 g/l MgSO₄.7H₂O; 1.5% agar(Becton Dickinson AG, Postfach, CH-4002 Basel, Switzerland) and traceelements (500-times concentrate: 5.0 g/l MnSO₄×1 H₂O, 2.0 g/lCOCl₂.6H₂O, 0.75 g/l (NH₄)₆Mo₇O₂₄.4H₂O, 0.5 g/l AlCl₃.6H₂O, 0.375 g/lCuCl.2H₂O)). Colonies were visible after 24 to 48 h. All transformantswere sensitive to neomycin indicating the replacement of the neomycingene by the introduced wild-type and mutated tkt genes.

Genomic DNA was isolated from the transformants and the tkt gene wasamplified by PCR as described in Example 1. The introduced mutationswere confirmed by sequencing. No further nucleotide exchanges wereobserved. The generated B. subtilis strains were called:

R357A-BS3403, R357H-BS3482, R357K-BS3484, R357G-BS3512, R357V-BS3487,R357I-BS3509, R357L-BS3507, R357T-BS3492, R357S-BS3490, R357M-BS3505,R357N-BS3486, R357Q-BS3488.

EXAMPLE 6 Transduction of B. subtilis RB50::[pRF69] (EP 0405 370) withBacteriophage PBS-1 Lysate of the Transketolase-Deficient Wild-TypeStrain BS3402

Transduction work with phage PBS-1 was done as described in Henkin etal., 1984 (Henkin, T. M., and G. H. Chambliss. 1984. Genetic mapping ofa mutation causing an alteration in Bacillus subtilis ribosomal proteinS4. Mol Gen Genet 193:364-9). For preparation of the PBS-1 lysate, thestrain BS3402 was grown on TBAB plates (5 μg/ml neomycin) at 37° C.overnight. The cells were used to inoculate 25 ml LB medium (BectonDickinson AG, Postfach, CH-4002 Basel, Switzerland) to an OD of Klett20-30 (using the green filter). When 50% of the cells were motile, 0.2ml of the PBS-1 phage lysate (Henkin, T. M., and G. H. Chambliss. 1984.Genetic mapping of a mutation causing an alteration in Bacillus subtilisribosomal protein S4. Mol Gen Genet 193:364-9) were added to 0.8 ml ofthe culture broth. After 30 min incubation at 37° C. under shaking, 9 mlLB-medium were added. This was followed by another 30 min incubationstep at 37° C. Then 4 μg/ml chloramphenicol were added, and theincubation was continued for another 2 hours. Finally, the tubes weretransferred into a 37° C. dry incubator where they were left overnight.On the next morning, the culture was filtered through a 0.45 μm filterand stored at 4° C. or directly used for transduction.

For the transduction of the riboflavin overproducing strainRB50::[pRF69], the strain was grown on a TBAB plate at 37° C. overnight.Cells of this plate were used to inoculate 25 ml LB-medium (Klett20-30). When the culture reached Klett 175, 0.8 ml of the cells weremixed with 0.2 ml of a PBS-1 phage lysate from strain BS3402 prepared asdescribed above. After 30 min incubation at 37° C. under shaking, cellswere spun down and suspended in 1 ml VY medium. This was followed by 1 hincubation under the identical conditions. 200 to 1000 μl of thetransduced cells were plated on a selection plate containing 2 μg/mlneomycin. Grown colonies were tested for neomycin resistance. After gDNAisolation (Example 1), a standard PCR using primer tkt 1S and Rec 2ASwas done to confirm the replacement of the tkt wild-type gene by theconstruct of Example 4. A confirmed strain was called BS3523.

EXAMPLE 7 Introduction of the Modified Transketolase Genes into StrainBS3523

For preparation of PBS-1 lysates of strains BS3403, BS3482, BS3484,BS3486, BS3490, and BS3512, the respective strains were grown on TBABplates (5 μg/ml neomycin) overnight at 37° C. Cells from those plateswere used to inoculate 25 ml LB medium to an OD of Klett 20-30 (usingthe green filter). When 50% of the cells were motile (around Klett 150),0.2 ml of the PBS-1 phage lysate (Henkin, T. M., and G. H. Chambliss.1984.

Genetic mapping of a mutation causing an alteration in Bacillus subtilisribosomal protein S4. Mol Gen Genet 193:364-9) were added to 0.8 ml ofthe culture broth. After 30 min incubation at 37° C. under slightshaking or turning (roller drum), 9 ml LB-medium were added to thecells. They were incubated for another 30 min under the same conditions.Chloramphenicol was added to a concentration of 4 μg/ml, and theincubation was continued for another 2 hours. The tubes were incubatedovernight at 37° C. without shaking. On the next morning, the culturewas filtered through a 0.45 μm filter and stored at 4° C. or directlyused for subsequent transduction. For this, the transketolase-deficientstrain BS3523 (see example 6) was grown on a TBAB plate overnight at 37°C. Cells from the plate were used to inoculate 25 ml LB-medium (Klett20-30). The culture was incubated at 37° C. under shaking. When theculture reached Klett 175, 0.8 ml of the cells were mixed with 0.2 ml ofPBS-1 phage lysate of each of the strains BS3403, BS3482, BS3484,BS3486, BS3490, and BS3512 as described above. After 30 min incubationat 37° C. under shaking, cells were spun down and suspended in 1 ml VYmedium. After 1 h incubation under the identical conditions, the cellswere spun down again, suspended in 0.2 ml 1×SMS medium and plated ontoselection plates (1×SMS as described above with 1 g/l glucose, 1 g/lSorbitol, and 15% agarose). Grown colonies were tested for loss ofneomycin resistance. After gDNA isolation (Example 1), a standard PCRusing primer tkt 1S and Rec 2AS was done to amplify the tkt gene fromthe genomic DNA. The tkt gene of colonies that showed a replacement ofthe inactivated tkt gene by an intact one, were sequenced to confirm theexistence of the mutations. The generated strains were called BS3525(BS3484 lysate), BS3528 (BS3482 lysate), BS3530 (BS3486), BS3534 (BS3403lysate), BS3535 (BS3490 lysate), BS3541 (BS3512 lysate).

EXAMPLE 8 Growth of the Transketolase Mutant Strains on Glucose andGluconate

To evaluate the effect of the transketolase mutations on viability andgrowth of B. subtilis, the maximal growth rate of the generated strainswas determined on 2 g/l glucose or gluconate. The following medium wasused: 1×SMS (2 g/l (NH₄)₂SO₄, 14 g/l K₂HPO₄, 6 g/l KH₂PO₄, 1 g/ltri-sodium citrate, 0.2 g/l MgSO₄.7H₂O), 2 g/l glucose or gluconate, 500μg/l yeast extract and trace elements solution as described in Example5. 25 ml of the described medium in a 300 ml flask with baffles wereinoculated from an overnight culture (5 ml VY, resuspended in 1 ml freshVY) to an OD of Klett 20 to 30. They were incubated at 37° C. undershaking (220 rpm). The OD of the cultures were followed in one hourintervals during the lag phase. During the logarithmic phase theinterval was reduced to 30 min. At least four data points during thelogarithmic phase were used for the determination of the maximal growthrate.

TABLE 1 B. subtilis Growth rate % wild Growth rate % wild mutant onglucose type on gluconate type ratio Wild type 0.480 100%  0.351 100% 1.42/1   PY79 R357G 0.384 80% 0.300 86% 1.28/0.93 R357S 0.372 78% 0.25472% 1.46/1.08 R357T 0.342 71% 0.240 68% 1.43/1.04 R357N 0.366 76% 0.23166% 1.58/1.15 R357A 0.381 79% 0.225 64% 1.69/1.23 R357L 0.324 68% 0.18954% 1.71/1.25 R357H 0.324 68% 0.174 50% 1.86/1.36 R357K 0.348 73% 0.17149% 2.04/1.48 R357I 0.243 51% 0.108 31% 2.25/1.65 R357Q 0.228 48% 0.0926% 2.53/1.83 R357V 0.297 62% 0.101 24% 2.94/2.58 R357M 0.258 54% 0.06619% 3.91/2.84 R357Y 0.222 46% 0.06 17% 3.70/2.71 R357F 0.174 36% 0.03811% 4.58/3.27 R357D 0.156 33% 0  0% —

The wild-type strain PY79 showed as expected the highest growth rate onboth substrates.

By introducing the different mutations at transketolase position 357,the growth on gluconate was, as expected, much more affected than thegrowth on glucose. Reduction of the maximal growth rate on gluconate wasused as a measurement for the effect of the transketolase mutation onthe flux through the non-oxidative pentose phosphate shunt and on theaccumulation of the pentose phosphates. A wide range of growth rateswere covered by the shown mutations.

EXAMPLE 9 Riboflavin Production in Shake Flasks

5 ml VY containing chloramphenicol (10 μg/ml) were inoculated with theriboflavin production strains RB50::[pRF69], BS32525, BS3528, BS34530,BS3434, BS34335, and BS3441 (see Example 7). After overnight incubation,the cells were spun down (15 min, 4000 rpm) and suspended in 1 mlscreening medium (2×SMS, 10 g/l glucose, 1 g/l yeast extract, and traceelements as described in example 5). A 200 ml flask with bafflescontaining 25 ml screening medium was inoculated with 0.25 ml of there-suspended cells. The cultures were incubated for 48 h at 37° C. in awater-saturated atmosphere. After 48 h incubation time, during which thesupplied glucose was used up in all of the cultures, a sample of 0.5 mlwas taken from the cultures, 35 μl 4 N NaOH was added and the mixturewas vortexed for 1 min. 465 μl 1 M potassium phosphate buffer, pH 6.8,was added directly afterwards. The mixture was cleared by 5 mincentrifugation at 14000 rpm (Eppendorf centrifuge 5415D). Thesupernatant was transferred into a new tube. Two different methods forriboflavin determination were used. For the calorimetric determination,200 μl of the supernatant was diluted with 800 μl water. The absorptionat 444 nm was multiplied with the factor of 0.03305 to obtain gramriboflavin per liter medium. For the final results, the obtained valueswere corrected for volume differences. The riboflavin concentration wasalso determined by HPLC according to Example 10. The results are shownin Table 2:

TABLE 2 HPLC UV results % of results % of Riboflavin RB50:: (riboflavinRB50:: Strain [mg/l] [pRF69] [mg/l] [pRF69] BS3528(R357H) 179 171% 139193% BS3535(R357S) 173 166% 136 188% BS3534(R357A) 144 138% 124 172%BS3525(R357K) 148 142% 112 155% BS3559(R357Q) 134 129% 103 143%BS3530(R357N) 126 120% 93 129% RB50::[pRF69] 104 100% 72 100%BS3541(R357G) 92  89% 66  92% BS3523(deletion) 90  87% 58  81%

Nearly all Bacillus strains containing a transketolase mutation showed aclearly increased riboflavin production, while the transketolasenegative strain produced less riboflavin than the control strain. In thecase of the R357H mutation, the riboflavin concentration was nearlydoubled.

EXAMPLE 10 Riboflavin Fermentation

Fermentation runs were performed as described in EP 405370.

Fermentations were run with strains (1) RB50::[pRF69], (2) BS3534(R357A), and (3) BS3528 (R357H). At 24 hours and 48 hours fermentationtime, concentrations of riboflavin and biomass (cell dry weight) weremeasured in the culture broth. As shown in Table 3, parent strainRB50::[pRF69] produced 9.8 g/l riboflavin in 48 h with a yield onsubstrate of 3.59% (w/w). Biomass was produced with a yield on substrateof 20.3% (w/w). Derivatives of RB50::[pRF69] expressing a modifiedtransketolase gene showed significant increases in riboflavinproduction. BS3528 and BS3534 produced 11.7 g/l and 14.6 g/l,respectively. This corresponds to a yield on glucose of 4.23% withBS3528 and 5.14% with BS3534, respectively (Table 3). These resultsdemonstrate that the modification of transketolase activity leads to anincrease in riboflavin productivity.

TABLE 3 Riboflavin and biomass yield on substrate after 48 hfermentation time B2 Yield Biomass Yield [%] (w/w) Difference [%] (w/w)Difference RB50::[pRF69] 3.59 ± 0.27 20.26 ± 0.80 BS3534 5.14 ± 0.09+43% 18.92 ± 0.37  −7% BS3528 4.23 ± 0.19 +18% 17.78 ± 1.73 −12%

EXAMPLE 11 Analytical Methods for Determination of Riboflavin

For determination of riboflavin, the following analytical method can beused (Bretzel et al., J. Ind. Microbiol. Biotechnol. 22, 19-26, 1999).

The chromatographic system was a Hewlett-Packard 1100 System equippedwith a binary pump, a column thermostat and a cooled auto sampler. Botha diode array detector and a fluorescence detector were used in line.Two signals were recorded, UV at 280 nm and fluorescence trace atexcitation 446 nm, emission 520 nm.

A stainless-steel Supercosil LC-8-DB column (150×4.6 mm, 3 μm particlesize) was used, together with a guard cartridge. The mobile phases were100 mM acetic acid (A) and methanol (B). A gradient elution according tothe following scheme was used:

Time [min] % A % B 0 98 2 6 98 2 15 50 50 25 50 50

The column temperature was set to 20° C., and the flow rate was 1.0ml/min. The run time was 25 min.

Fermentation samples were diluted, filtered and analyzed without furthertreatment.

Riboflavin was quantitated by comparison with an external standard. Thecalculations were based on the UV signal at 280 nm. Riboflavin purchasedfrom Fluka (9471 Buchs, Switzerland) was used as standard material(purity≧99.0%).

1. A microorganism selected from the group consisting of Bacillus sp.and Corynebacterium glutamicum genetically engineered with apolynucleotide encoding a modified transketolase having an amino acidsequence containing at least one mutation corresponding to a mutation atposition 357 as shown in SEQ ID NO:2, wherein the polynucleotideencoding the transketolase is shown in SEQ ID NO:1, and wherein themicroorganism's growth rate on a carbon source that is metabolizedexclusively by the pentose phosphate pathway is reduced between 10 to90% in comparison with a non-genetically engineered microorganismcontaining a non-modified transketolase and wherein thegenetically-engineered microorganism is prototrophic for aromatic aminoacids.
 2. The microorganism according to claim 1 wherein thetransketolase is a Bacillus transketolase.
 3. A process for productionof a fermentation product for which ribose-5-phosphate,ribulose-5-phosphate, or xylulose-5-phosphate is a biosyntheticprecursor, the process comprising: (a) culturing a microorganismselected from the group consisting of Bacillus sp. and Corynebacteriumglutamicum genetically engineered with a polynucleotide encoding amodified transketolase having an amino acid sequence containing at leastone mutation corresponding to a mutation at position 357 as shown in SEQID NO:2, wherein the polynucleotide encoding the transketolase is shownin SEQ ID NO:1, and wherein the genetically-engineered microorganism'sgrowth rate on a carbon source that is metabolized exclusively by thepentose phosphate pathway is reduced between 10 to 90% in comparisonwith a non-genetically-engineered microorganism containing anon-modified transketolase and wherein the genetically-engineeredmicroorganism is prototrophic for aromatic amino acids, in a suitablemedium under conditions that allow expression of the modifiedtransketolase and biosynthesis of the fermentation product; and (b)separating the fermentation product from the medium.
 4. The processaccording to claim 3 wherein the fermentation product is selected fromthe group consisting of riboflavin, a riboflavin precursor, FMN, FAD,pyridoxal phosphate, and one or more derivatives thereof.
 5. The processaccording to claim 3 wherein the fermentation product is riboflavin. 6.A process for production of a fermentation product, wherein thefermentation product is selected from the group consisting ofriboflavin, a riboflavin precursor, flavin mononucleotide (FMN), flavinadenine dinucleotide (FAD), pyridoxal phosphate, and one or morederivatives of the aforementioned compounds, in a microorganism selectedfrom the group consisting of Bacillus sp. and Corynebacterium glutamicumhaving a modified transketolase, wherein the amino acid sequence of thetransketolase contains at least one mutation corresponding to a mutationat position 357 as shown in SEQ ID NO:2, wherein the polynucleotideencoding the transketolase is shown in SEQ ID NO:1, so that the specificactivity of the modified transketolase is modulated in comparison to thecorresponding non-modified wild-type enzyme, and wherein themicroorganism is prototrophic for aromatic amino acids, the processcomprising: (a) culturing said microorganism in a medium containingglucose or gluconate as a carbon source under conditions that allowexpression of the modified transketolase and biosynthesis of thefermentation product and (b) isolating the fermentation product.
 7. Theprocess according to claim 6 wherein the fermentation product isriboflavin.
 8. The process according to claim 3 wherein thetransketolase is a Bacillus transketolase.
 9. The process according toclaim 6 wherein the microorganism's growth rate on glucose or gluconateis reduced between 10 to 90% in comparison with anon-genetically-engineered microorganism containing a non-modifiedtransketolase.